Free radicals and related activated
electronic species are produced in biological systems in
antimicrobial defense, through the action of the mixed function
monooxygenases, by various oxidative enzymes such as xanthine
oxidase, and by autooxidations mediated by such agents as heavy
metals or quinones. While the evidence is circumstantial, £xcessive
unconfined or inappropriate production of radical species in
inflammation, the metabolism of exogenous chemicals, or through
autooxidation probably plays a significant role in human disease.

Introduction.

Free radicals are short-lived reactive
chemical species having one or more electrons with unpaired spins.
Previous authors (1-44) have extensively reviewed the evidence for
roles for such electronicallyactivated species (e.g., superoxide and
singlet oxygen) in the normal function of cells and tissues and in
the etiology of certain diseases in man. Radical generating processes
may be key components in the toxicity of many drugs,
(8,9,11,20,22-28.34,42,47-52) in antimicrobial defense,
(5,6,10,34,38-46) and in inflammation.(7,32,41) This review,
restricted to "acute" pathologic processes such as
inflammation and drug toxicity, broadly summarizes our current
understanding of this field, its seductive and circumstantial nature,
and its clinical relevance. Since an in-depth analysis of any of the
individual areas of this field could amount to a book in itself, we
have made certain other arbitrary limitations on this review.

For example, for the roles of free
radicals in aging and in carcinogenesis, the concerned reader is
referred to other authors, while Table I lists a few events relevant
to the role of active mechanisms in human disease. Likewise, to cover
such broad areas comprehensibly in a brief review requires that the
reader refer to previous authors (l-44) for more detailed analysis
of each individual area. Hopefully, the resulting broad synthesis
justifies such liberties.

The Nature of Radical
and Excited-state Species. (See Glossary).

Moving charges generate magnetic
fields. An orbital electron may be viewed as a moving negative charge
which generates a magnetic field ("magnetic moment''). Depending
on the direction of its motion ("spin") about the orbital
axis, this magnetic field will have its north magnetic pole oriented
either "up" or "down" relative to its orbit.
Logically, this magnetic property of the electron is called "spin"
(Figure 1).

Stable compounds, especially those
composed of low atomic weight atoms, have even numbers of electrons
arranged two to each "orbital". The two electrons in each
orbital are "paired". That is, their "spin"-derived
magnetic fields are of opposite polarity, effectively canceling them.

A free radical with one or more of its
electrons unpaired has either an odd number of orbital electrons,
with one unpaired - a free radical per se - or of pairs of
electrons of the same spin isolated singly in separate orbitals.
Molecular oxygen is an example of such a biradical. (16,17) These
uncancelled spins give radical species a net magnetic moment which
can be directly detected by electron spin resonance (ESR)
spectroscopy, a technique which involves aligning the unpaired
electron with an external magnetic field and then measuring its
relaxation as the field is removed.(23,27,28), A compound may become
a free radical either by gaining (e.g., superoxide radical) or by
losing (e.g., ascorbate radical) a single electron in a reduction/
oxidation reaction. Such a reaction produces the radical products of
both molecules, and can result in radical chain reactions.(16-19) The
rate of propagation of radical chain reactions depends upon the
reactivity of the radicals formed with the substrates
available.(15-21), Figure 2 shows an example of such a chain
reaction, the oxidative degradation of unsaturated fatty acids.

In biological systems radical species
tend to be restricted to derivatives of molecular oxygen (Figure 3),
polyunsaturated fatty acids (Figure 2), sulfhydryl compounds,
quinones or quinone-like compounds including flavins (Figure 5) and
other compounds which can easily transfer single electrons.

Free radicals are generally highly
reactive and participate in hydrogen abstraction, radical addition,
bond scission, and annihilation reactions. For example, they can
oxidize unsaturated fatty acids in cell membranes,(15-21( damage DNA,
(53-57) oxidize protein amino acid side-chains,(16-19,58,59)
depolymerize hyaluronic acid,(60) modulate nucleotide cyclase
activities,(61-67) and the action and synthesis of prostaglandins and
lipoperoxides.(48,68-71). In short, radical species can attack most
biological substrates from large macromolecules to smaller molecules
such as catechols. Exceptionally, stable radical species can be
formed, mostly due to steric protection of the unpaired electron.
Examples include the melanins (72-75)and the stable radical
derivatives produced in spin-trapping measurements.(7,28)

The high reactivity of free radicals
and active oxygen species virtually precludes direct detection in
organs and tissue by electron spin resonance techniques. In order for
specific species to be detected in aqueous systems, micromolar
steady-state concentrations are required.

Such levels are rarely reached in vivo
.since the life span of radical species is usually extremely short, I
msec. or less. Other authors (27,28) have reviewed the various
techniques for the detection of radical and related species in
biological systems. Table II outlines some of these methods: Such
techniques may take advantage of the relative specificity of such
enzymes as SOD or catalase toward active species of oxygen, involve
the inhibition of a process by antioxidants or measure production of
specific reaction products.

Radical production can also be measured
by means of "spin-trapping" techniques, in which a
biologically-produced short-lived radical oxidizes another compound
to form a long-lived (and thus detectable) radical derivative.(27,28)

Active Oxygen System.

While other radical species may be of
importance in such processes as drug
metabolismll.(15,20.24-30.34,42,4755.76-89) and in radical chain
reactions,(8-19) radical forms of oxygen are the penultimate species
in aerobic systems. Figure 3 presents a simplified outline of the
major steps in the four-electron reduction of oxygen to water, which
occurs through the addition of single electrons to oxygen within the
cell by enzymes or by simple chemical reducing agents. (1-42,90-93)
Other, less-defined, species (such as hydroperoxides) may also be
present.

Mitochondria carry out the entire four
electron reduction of molecular oxygen to water at cytochrome
oxidase. Likewise, the cytochrome P-450 centered mixed function
monooxygenase of liver endoplasmic reticulum - and of other tissues -
reduces molecular oxygen to active metabolites, which it uses to
oxidize a wide variety of substrates.

Although the chemistry involved is
beyond the scope of this review [for further information the reader
is referred (034.81-83.92], the reaction occurs by a series of
single-electron transfer steps. The products include free radical
intermediates, which are subsequently either reduced further or
released from the active site of the enzyme and react with
nucleophilic groups in the immediate molecular environment -
including molecular oxygen.(8,9.11,15.24,27.28.30) Similarly,
xanthine oxidase4 oxidizes products of purine catabolism as well as
other oxidizable substrates with the generation of reactive oxygen
metabolites, while vitamin K-dependent carboxylation of clotting
factors in liver also involves generation of active oxygen
species(93).

The NAD(P)H oxidase ("superoxide
synthetase")/ myeloperoxidase system of inflammatory cells
(5,6,3l-33,3S-40,46,94-106) is also of g!eat importance. This
membrane-bound system, which is activated by the phagocytosis of
foreign materials such as bacteria by phagocytic white blood cells,
generates large amounts of superoxide and other microbiocidal and
cytocidal oxygen derivatives (Figure 4). Similar systems may also be
present in T -lymphocytes, 96 platelets, 10,97 conjunctival mucus
(98) and adipocytes.(61). Electrons may also be added directly to
molecular oxygen in "autooxidation" reactions such as those
involved in the typically metal-catalyzed direct transfer of an
electron from a reducing substrate to molecular oxygen or peroxide
without an enzyme mediator. (16-21,24,25,52,107-1 17) Reducing agents
participating in such reactions include catechols, . .d 24 lfh d I d
24107-109 unc aCl, su y ry compoun s, , quinones, 110 metals/4,3s,1
10-1 16 halides/4 alpha and beta chains of hemoglobin,Il6,117
ascorbate/4,S7,114,IIS paraquat,44,63 and anthracycline drugs such as
adriamycin.2o,2s,42,sl If the oxidized derivative is then re-reduced
by some other agent such as NADPH, a process known as "redox
cycling" occurs. Such a mechanism has been proposed to explain
the toxicity and action of quinone derivatives of adriamycin
[reviews/O,2S,42,SI] which may shuttle electrons between the
hexosemono-phosphate [HMP] shunt and molecular oxy-gen (Figure 5).

Singlet Oxygen and other Excited
Molecules. An excited-state species is produced when an orbital
electron absorbs energy

FIGURE 3.

The active oxygen
system. In biologic systems molecular oxygen is reduced to
water in four oneelectron steps. Reduction of molecular oxygen to
superoxide, and of peroxide to hydroxyl radical are "spin
forbidden" and thus are slow unless catalyzed by a heavy ion.
Alternative spin-permitted pathways for the reduction of 02 include
interaction of molecular oxygen with the excited triple state of
another molecule to produce singlet oxygen (from light (7,91,118,119)
or an excited state molecule (99120121) and jumps to a higher energy
orbital on the same atom. Excited-state species produced may be
highly reactive and participate in reactions not unlike those of free
radicals.(91,99,119-122) Singlet oxygen (Figure 2), itself a strong
oxidizing agent, may be responsible for some of the effects assigned
to other active oxygen species such as superoxide.(91,122) Roles for
singlet oxygen have also been postulated in photosensitized
reactions,(90,91,113,119) and in antimicrobial defense.
(5,6,33,46.99, 105,120).

In biological systems the sources of
the electrons are generally enzymes (e.g., NAD(P)H oxidase) and
reducing substances (electron-donors). Simplistically,
electron-donors act as antioxidants by (e.g.) reducing more reactive
species such as trichloromethyl, superoxide, or hydroxyl radicals to
less reactive species such as chloroform, peroxide, or water.
Conversely, electron-donors act as pro-oxidants by reducing less
reactive species such as molecular oxygen and peroxide to more
reactive species via reactions which are typically mediated by the
cyclical reduction/oxidation of transition-metal ions. The
reduction of peroxide to hydroxyl radical by ferrous iron is known as
Fenton's reaction."2 Peroxide and superoxide can also react in
the presence of a metal ion to produce hydroxyl radical and molecular
oxygen. This latter reaction is called the "Haber Weiss
Reaction". (111-115).

Cellular Defense
Against Radical Species.

The ubiquity and reactivity of
radical-generating systems in cells has resulted in the evolution of
defense mechanisms against the damaging effects of such powerful
oxidizing agents.(4,12,16-19,21,36,123-128) Figure 6 summarizes the
activity of these intracellular enzymes.SOD (436 125 126). Superoxide
dismutase and catalase (1-4,126) catalyze the dismutation of
superoxide and hydrogen peroxide, respectively, .GSH peroxidase
reduces hydrogen and organic peroxides (123,124,126) to water and
alcohols, respectively. GSH S-transferases (27) act by transfering
glutathione residues to electrophilic reactive metabolites of
xenobiotics.

Oxidized glutathione (GSSG) produced is
rapidly reduced by reactions utilizing NADPH generated from various
intracellular systems, including the hexose-monophosphate
shunt.(123,124). Various organelle specific isoenzymes of superoxide
dismutase exist. (4,125). The Zn, Cu SOD is cytoplasmic, while the
Zn, Mn enzyme is chiefly mitochondrial. Neither isoenzyme is found in
high concentrations in extracellular fluids. (4,36,125,129).

Many reducing agents (Table III) also
serve as antioxidants, reducing the more active

radical species such as peroxy or
hydroxyl radicals (1,3, 14-21,24,58, 130-134) to less reactive forms
(e.g., water) (Figure I), as well as de-excite singlet
oxygen.(ll9,l35) These compounds also terminate radical chain
reactions.(14-21,24).

However, chemical antioxidant defense
is a double-edged sword. First, when a reducing agent scavenges a
radical, its own radical derivative is formed. Thus, unless the
radical is extremely stable, a radical chain reaction may continue.
Second, as implied in Figure 2, a reducing agent may itself reduce
oxygen to superoxide or peroxide to hydroxyl radical in autooxidation
reactions(20,24,107-117. For example, ascorbate (57,114) and uric acid
(4) which may functlion as antioxidants, parhcIpate in
autooxidations, either directly, by reducing some other oxygen
activator such as a transition-series metal or quinone, or by acting as an enzyme cofactor (19,24,57,58,107-117)

FIGURE 6. Antioxidant
defense - enzymes. Three important intracellular enzymes
constitute antioxidant defense; superoxide dismutase (SOD), catalase,
and the GSH peroxidase/GSSG reductase system. SOD catalyzes the
dismutation of superoxide, catalase the conversion of hydrogen
peroxide to H2O and O2, while GSH peroxidase transfers electrons from
GSH to reduce peroxides to water. The oxidized glutathione produced
(GSSG) is re-reduced back to GSH by glutathione reductase utilizing
NADPH produced by the HMP shunt.acting as an enzyme
cofactor(19,24,57,58,I07-117.

Such processes may account for
ascorbate's ability to depolymerize DNA,(57) inhibit brain N a + / K+
ATPase, (114) potentiate the toxicity of paraquat,(136) and mediate
lipid peroxidation.(1l4) They may also contribute to the
pathophysiology of disorders of purine metabolism. (24,26,137,138).
The exact mix of pro or antioxidant properties for a particular
reducing agent is a complex integration of several factors. In the
case of hydroxyl radical scavengers, the product of radical
interaction with the antioxidant is generally much less reactive than
hydroxyl radical, one of the most powerful oxidizing agents known.
Or, the radical forms may be sufficiently stable and present in high
enough concentration that they self-annihilate - as is the case with
glutathione and superoxide. Similarly, pH greatly influences the
direct reduction of oxygen to superoxide by sulfhydryl compounds,
(107) while other local factors such as the molar concentration of
oxygen molecules are also important.

Excited-state derivatives such as
singlet oxygen and the excited triplet (diradical) states of other
molecules may be quenched by interactions with conjugated diene
systems such as those found in carotenes,(1,19), tocopherols,(135),
or the melanins. (24,26,45, 73-79). As is the case with reducing
antioxidants, such compounds may also produce active electronic
species (45,73-79,118) and perhaps disease.

In addition to environmental causes
such as oxygen(4,13,29,139,147), light (90,91), or ionizing radiation
(53,148-149) three physiological circumstances result in
extraordinarily high local fluxes of radical species: (1) activation
of the P-450-centered mixed function oxidase systems of endoplasmic
reticulum, (2) activation of NADPH oxidase in phagocytes in response
to antimicrobial defense and inflammation (Figure 4) and (3) the
presence of extraordinarily high levels of compounds which can reduce
oxygen directly in autooxidation reactions.

Under such circumstances, the rate of
active species generation may exceed the local capacity of the
antioxidant defense and may contribute to injury.

However, before we proceed we must
again insert a note of caution. With the exception of melanin, the
radical species present in biological systems are very short-lived
and are present at such low concentrations that they are usually not
detectable in vivo by electron spin resonance spectrometry.
Indeed, they may be detected in vitro only under very special
conditions (27,28). Thus, evidence for the involvement of any radical
or excited-state species in a particular biological process (much
less in a human disease) is necessarily indirect, often
circumstantial and must be taken cum grano salis (with a grain
of salt). Nonetheless, multiple bits of evidence (which taken
separately are inconclusive) collectively strongly support a role for
electronically-activated species in at least some processes such as
inflammation and in the toxicity of some drugs, although the exact
role and identity of such species is a subject of much controversy
(e.g. 150,151).

Table IV outlines some criteria for
acceptance of a role for free radicals in specific human diseases.
These criteria include such things as inhibition by a presumably
specific enzyme such as SOD or the detection of specific reaction
products. For example, Wilson's Disease (with high local tissue
copper contents, and lower levels of ceruloplasmin, a copper
containing protein which may function as an extracellular
antioxidant/I,128) qualifies as a putative radical-associated disease
under criteria I and IV. Yet, to the best of our knowledge, no one
has yet demonstrated an accelerated flux of superoxide, increased
products of lipid peroxidation or other free radical metabolites in
this disease. Further application of these criteria to other disease
processes is an exercise for the critical evaluator to ponder.

TABLE IV. Criteria for
involvement of radical processes.

I. The condition is known to be
associated with abnormal production of free radicals and other
electronically activated species (e.g., granulocyte activation in
inflammation, or the presence of a putative charge-transfer agent in
abnormal amounts or forms - such as copper in Wilson's disease).

II. Demonstration of specific radical
species or their unique reaction products at the site of a lesion.

III. In vitro demonstration that
radical species are involved in important mechanisms relevant to the
disease in question.

IV. Production of similar symptoms and
lesions by otherwise dissimilar chemicals which produce free radical
species in common, or which inhibit or deplete components of the
natural antioxidant defense system (the "common symptom"
test).

V. A corollary to IV: the ability to
modulate the pathophysiologic progression of a disease
pharmacologically through the intervention of ectopically
administered SOD, catalase, antioxidants, or free radical quenchers.

Inflammation.

The enzymatic production of active
oxygen species by inflammatory cells (Figure 4) may contribute to the
pathophysiology of leucocyte dependent inflammatory processes
[reviews l,7,16,31-33]. As outlined in Figure 4, in addition to their
direct action on cellular constituents
(1,7,16,31-33,38-41,96,100,152) oxygen metabolites may also act as
specific modulators of the inflammatory process. For example, in
vitro active oxygen species can affect the activity of inflammatory
immunomodulators such as interferon (153) leucocyte-dependent inflammatory processes (Reviews, 1,7,16,31-32)
(154-156), leucocyte clastogenic factors (154-156) , lymphocyte clastogenic
factors(157-159), soluble immune response suppressor (160), serum
protease inhibitors (161-164), and vascular
permeabi1ity-regulating factors.(165,166). Similarly, extracellular
active oxygen species may also directly influence platelet (l67-I71)
and fibrocyte (63,172) function. They may also be involved in the
metabolism and action of such important modulator substances as the
prostaglandins (41,68) leucotrienes,(17I), unsaturated fatty acids
(41,68-71) and cyclic nucleotides (61-67). They may also influence
interactions between lymphokines and macrophages (l56,173) and
lymphocyte function (l74) as well as induce histamine release from
mast cells (175). The specificity of such effects suggests that
active metabolites of oxygen may be acting more as messenger
substances than as non-specific chemical reagents. (emphasis-added).

Dr P notes: Some history-- We first proposed the global cellular messenger
role of electronically-activated species at 1979 meeting of free radical
investigators in Honolulu. It is now
generally accepted and is dubbed "redox signaling". There is even a
journal of that name. But, at the time, an outward statement
of the concept was viewed with significant scholarly hostility and could not
get past reviewers. So I snuck it in here --evidently
nobody noticed or perhaps the time was ripe. Anyway,
even delayed five years, this seems to be the first
notation in the published literature. Normally, I think
priority disputes are (well) tacky. But hopefully in view of the events described in
www.organic semiconductors.com,
I can be forgiven this brief flash of ego.
.

Such circumstantial in vitro evidence
hould be assessed with the special caution reserved for this
difficult field. Nonetheless, taken globally, it is not surprising
that antioxidents (l76-183), SOD (1,138,154,165,177,184-194) and
catalase (32,177,195) should ameliorate inflammatory symptoms in
human and animal systems.

This is of some clinical importance,
since an antiinflammatory pharmaceutical preparation rich in SOD
("orgotein") is used in veterinary medicine and recently
has been shown to be both effective and apparently safe in the
treatment of various inflammatory lesions in man.
(1,138,154,165,177,184-194). Catalase has also been used in the
treatment of arthritic disease in man with reported success. (195).
Likewise, the action of such established antiflammatory drugs as the
corticosteroids, (196) penicillamine,(197) and many non-steroidal
antiinflammatory agents (1,76-183,198-200) may be at least partially
dependent upon interference with active oxygen metabolism in
phagocytes.

Further, peroxidase release from
eosinophils may playa similar role in inhibition of the inflammatory
response (201) while the antioxidant properties of ceruloplasmin may
also give this compound antiinflammatory properties.(21,128).

An illustrative (albeit circumstantial)
model for the role of superoxide in rheumatoid arthritis can be
postulated as follows: Granulocytes tend to be concentrated at sites
of active rheumatic disease, presumably in response to the presence
of immune complexes and other immunomodulator substances. Superoxide,
peroxide, and other active oxygen species produced further kindle the
inflammatory process by specific mechanisms such as those outlined in
Figure 4. Catalysis of radical oxidations by transition-sernies
metals may also play a role. Agents such as ectopic SOD may interfere
with this process by destroying active oxygen species or increasing
peroxide fluxes, thus interfering with one or more of the mechanisms
detailed above and in Figure 4.

Since SOD is one of the most
substrate-specific enzymes known, at first sight its efficacy in
rheumatoid arthritis and other lesions strongly implies a role for
active oxygen species in such diseases and a role for
superoxide-destroying agents in their treatment. However, there are
serious problems with such an assumption. For example, the course of
action of SOD as an inflammatory agent often bears no apparent
relationship to its serum levels (1,185) and the "denatured"
enzyme still possesses significant antiinflammatory properties. 185
Feel (51) even ably questions the specific role of the protein in
destroying superoxide.

With this caution, active oxygen
species may play a significant role in the etiology of other
inflammatory lesions in man, For example, SOD is reported to be
effective in the treatment of lupus erythematosis,(43,158) and unique
light-activated, superoxide-dependent lymphocyte clastogenic factors
present in the serum of patients with lupus and other collagen
diseases may account for some of the photosensitivity of this group
of disorders.(157-159). Both direct and indirect production of active
oxygen species may also have a role in the pathophysiology of gout
and other hyperuricemic syndromes.(24,26,173,138,203,204). For
example, urate, a reducing agent, is present in the extracellular
environment at concentrations approximating 0.3 mMolar (130). Like
many reducing agents, it apparently has both antioxidative (3O-134) and
autooxidative (24) properties. In fact, it may have taken over some
of the functions of ascorbate in primates.(133). Likewise, xanthine
oxidase is an effective producer of oxygen radical species, while
urate itself stimulates the production of active species of oxygen by
phagocytes (203,204) and may protect cycloepoxigenase from
autooxidation.(131) The effectiveness of SOD in the treatment of
urate-induced inflammatory disease in Dalmations suggests a role for
superoxide in this lesion.(138).

Production of active oxygen species by
activated phagocytes may also have a role in vascular (and other)
damage following endotoxin shock,(144,145,205), burn-induced plasma
volume loss (71) and even in atherosclerosis.(205). Similar
mechanisms may account for the possible role of radical species in
the progression of damage following neuronal
injury.(14,17,56,171,206). Antioxidants such as the methoxyphenols
are apparently effective in the amelioration of both experimental
cerebral edema and spinal cord injury.(17,56). However, once again we
must emphasize that, like most else in this field, the evidence for
free radical involvement in inflammation and neuronal injury is
circumstantial and has not been proven conclusively.

Radical Mechanisms in
Drug Toxicity.

Oxidation of chemicals by the p450
system generates free radical and other metabolites such as epoxides
and aldehydes which may interact with cellular constituents.
(8,9,11,15,1721,27,28,30,34,73,80-86,110,207,208) F I t. Forr
example, partly as a result of recent spin-trapping studies,(8,9,73)
perhaps the best established role of free radicals per se is
in the toxicity of halogenated hydrocarbons such as carbon
tetrachloride and halothane. (8,9, 11,28,30).. Nonetheless, (typical
for this field) it remains unclear whether it is (e.g.) the
trichloromethyl radical, peroxide, or the peroxy radical or its
reaction products with oxygen which cause injury in carbon
tetrachloride poisoning.

Thus, the toxicity of acetaminophen may
be due to its oxidation to an iminequinone, which in a reduced state
may either interact with vital nucleophilic targets within the cell,
or generate superoxide anion, or both.(81,85), Similarly, the
antibacterial and toxic potential of 5-nitrofurantoin appears to be
related to its reduction to an anion radical, which among other
alternatives can generate superoxide anion.(83). Similar mechanisms
have been proposed to account for toxicity of
chlorpromazine.(27,28,77,80). Also, the phototoxicity of many agents
appears to depend upon the production of singlet oxygen in the
presence of an excited triplet state produced by the action of light
on the photosensitizer.(37,90,91).

Such reactions follow similar pathways
to ultimately result in the reduction of oxygen to water.
Light-induced superoxide production may be responsible for the
photosensitivity found in lupus and other collagen diseases.
(157-159), Direct, nonenzymatic reduction of oxygen to active species
may have a role in both the action and toxicity of the antitumor
agents adriamycin (I,20,22,25,42,51,52,67), bleomycin (22,49,51,
53b-55) and cis-platinum.(1,49.51). In vitro, both . .adriamycin and
bleomycin' (otherwise totally-dissimilar drugs) bind to DN A and
damage it by active-oxygen dependent mechanisms and may participate
in redox cycling.(20,42)

Likewise, their toxicity in .many
systems, and that of cis-platinum, (1,49,51,209) can be
ameliorated by the administration of ectopic SOD and/or catalase.
Vitamin E ameliorates the toxicity of adriamycin (5) but only if
given before administration of the antitumor agent.(210),
Significantly (although it may not be generally true in man (211),
many animal tumors appear to be deficient in defense mechanisms
against radical species relative to normal cells (review, 22). This
may increase the relative antitumor specificity of radical-generating
antitumor agents(22).

Since SOD and catalase (which probably
do not penetrate into cells) ameliorate the toxicity of such agents
in vivo, a significant fraction of their toxicity may be due
to extracellular production of oxygen metabolites, in line with the
relative paucity of defense mechanisms in the extracellular space. In
support, adriamycin is cytotoxic in vitro when bound to an insoluble
extracellular substrate.(212,213). In turn, this holds out the
possibility that the (putative) intracellular antitumor activity of
such drugs (say, at the DNA level) may be separable from that
fraction of their toxicity which is extracellular. SOD apparently
does not diminish the antitumor effect of such drugs.(1,51). However,
the situation is confused by the apparent partial antitumor activity
of SOD itself, (43,51,215) which may happen with the "denatured"
enzyme,216 and which is roughly equivalent to cis-platinum in at
least one experimental animal tumor system. (57).

Active Oxygen and
Fibrosis.

Oxygen in high concentration produces
diffuse alveolar damage (DAD), often progressing to interstitial
pulmonary fibrosis, which is essentially identical to that caused by
a myriad of other causes, including drugs, endotoxins, thermal .
.injury, and infammatory processes, , , ,(146,217,218), many of which
may involve production of active oxygen metabolites. The toxicity of
oxygen itself is likely due to the productlon of active oxygen
species, while the radical-producing antitumor agent .bleomycin
produces pulmonary injury and subsequent interstitial pulmonary
fibrosis.(146). Further, a variety of other radical generating agents
are associated (perhaps fortuitously) with similar alveolar damage
and/ or fibrotic symptoms. Examples include the pulmonary lesions
induced by such agents (44, 219) as paraquat,' mtro furantoins, or
bleomucn and the vitreous fibrosis induced by interocular hemoglobin
or elemental copper. (220).

Significantly, ectopic SOD (221) and
catalase (222) apparently give some protection in experimental
paraquat poisoning, although this has not as yet been borne out in
human trials.(44). Likewise, SOD has been reported to be effective in
such fibrotic lesions as Peyronies disease (l89) and
radiation-induced cystitis.(148,188). Superoxide is reported to
activate fibrocyte function in vitro, stimulating such processes as
collagen production (l72) and guanylate cyclase.(63).

We speculate that the stimulation of
(e.g.) the fibrotic response by these radical-generating agents may
be a consequence of their common interaction with existing modulator
mechanisms coupling production of active oxygen species by
inflammatory cells with fibrocyte activation, collagen production,
and wound healing as part of the inflammatory response.

Other Charge- Transfer
Agent Associated Diseases.

The chronic presence of elevated levels
of other autooxidation-catalyzing charge-transfer agents such as
copper or manganese is associated with human disease states, although
it is again an open question whether radical mechanisms contribute to
the pathophysiology of such diseases. Nonetheless, some clinically
useful (if speculative and unproven) associations have been made.

For example, the conjectures of Cotzias
et al.(76.223) concerning the possible role of a metal/
neuromelanin/ free radical interaction in the pathophysiology of
manganese poisoning, Wilson's Disease, and phenothiazine toxicity
were one of the major factors leading to the trial of levo-dopa in
manganese poisoning and, ultimately, Parkinson's disease.(223)
Similar considerations concerning inner ear melanin, drug-induced
deafness, and nephrotoxicity prompted the use of SOD and catalase in
the amelioration of cis-platinum toxicity.(49.5l). Too broad an area
to consider here in detail, various aspects of this field have been
the subject of work by other authors e.g,,
(24.26.45,75-80,89,223-250).

Briefly, ionic forms of copper, iron,
and manganese (as well as other "charge transfer" agents)
can all catalyze radical oxidations in vitro. The respective
diseases associated with chronic elevated systemic levels of such
compounds (e.g., Wilson's disease, hemochromatosis and chronic
manganism) are variably associated with one or more of a set of six
specific symptoms. These symptoms may include movement disorders,
"psychosis", pigmentary abnormalities, deafness, fibrotic
lesions such as cirrhosis, and arthritic symptoms. For example,
hemochromatosis, an iron storage disease, may present with most or
all of these symptoms (vis, the role of iron in catalysis of radical
oxidations (3,53-55,92,lll-115).

As we have seen, the last two symptoms
(fibrosis and arthritis) may be referable to radical mechanisms under
other conditions (Figure 4), while several putative
neurotransmitter-dependent processes (such as burn-induced plasma
volume loss,(71) oxygen, (139,143) or adriamycin toxicity (51)
serotonin release from platelets (168), histamine release from mast
cells,(175) and autocoid-induced edema in mice (l65)) may be
modulated by active species of oxygen. Direct oxidative pathways for
neurotransmitter metabolism are also possible
(4,84,88,89,114,244,246-250) as are other effects on neurotransmitter
function. (17,24,26,45,49,89,1 14,143, 223,249).

The same associations between
pigmentary abnormalities and other specific symptomatology appear to
hold for other "chargetransfer" agent associated diseases
such as alcaptonuria, hyperruricemic syndromes, homocystinuria, and
chronic iodism.(24,26,138,235). Further, both drug-induced cutaneous
photosensitivity (a process which likely involves generation of
active oxygen species (90,91)), and the clinical use of such
oxygen-radical-generating antitumor agents as bleomycin and
adriamycin (253) are commonly associated with hyper-pigmentation.
While it is not within the bounds of this review to consider such
associations in depth, many charge-transfer agents bind readily to
melanin (229,230) for much the same reason that they bind to
molecular oxygen to catalyze autooxidations (24,26). Further, since
melanin may be a protective agent against active species, the common
presence of pigmentary abnormalities in such diseases and in
inflammatory and photosensitivity diseases (e.g., Lupus
erythematosus) may be an outward reflection of some ongoing
electronically-activated process. Once again, such circumstantial
evidence must be approached with caution.

Interestingly, pigmentary abnormalities
alone may be associated with specific neurological symptoms such as
deafness and movement disorder.(26). For example, the association
between pigmentary abnormalities and deafness in such diseases as
Waardenburg's and Usher's syndromes is a commonplace in audiology
(228-232) and may be present in over 10% of cases of severe
congenital deafness.(231). Likewise, the toxicity of such oxotoxic/
nephrotoxic drugs as kanamycin (26,45,47,49) and cis-platinum
(49,51,149) may be due to free radical mechanisms and / or to binding
to inner ear melanin.(229,230) Similar mechanisms may hold for the
nephrotoxicity of agaricus bisporus toxin (254) and
para-aminophenol,(110) as well as the renal toxicity of
radical-producing agents such as adriamycin or paraquat.

Enzyme Deficiencies.

The various protective mechanisms are
usually adequate to protect biological systems against normally
present active species except under conditions of high radical load.
However, in glucose-6phosphate dehydrogenase (G6PD) deficiency the
activity of the GSH reductase/ peroxidase system of the red blood
cell is greatly curtailed secondary to nonregeneration of NADPH
(Figure 6). The red cell is particularly subject to radical load
because the hemoglobin and its side-chains may be generators of
active oxygen species (117).

Normally, even in the absence of G6PD,
the remaining activity of the GSH reductase/ peroxidase system in
concert with the other protective mechanisms is adequate to protect
the red cell. However, under conditions of oxidative load as may
occur in antimalarial treatment, they are inadequate, resulting in
hemolysis. (101,124,255). Acatalasemia in humans has minor
symptomology (sterile mouth ulcers (256), perhaps due to the
intactness of other defense mechanisms. Similarly, a relative
deficiency of granulocyte superoxide dismutase has been reported in
juvenile rheumatoid arthritis.(257) Deficiency of the active-oxygen
generating NADPH oxidase system in granulocytes is present in chronic
granulomatous disease (7,8,13) and may be associated with sepsis in
burn injury.(258).

Summary and Clinical
Implications.

In summary, we must reiterate a few key
points. First, most evidence for active radical mechanisms in
biological processes and in human disease states is circumstantial.
At present we are unable to measure such species directly in man.
Clearly, as our understanding of the problems increases, better
techniques for the study, diagnosis, and moderation of such processes
should develop. Second, protective mechanisms suffice to protect
organisms against such species under normal conditions and are
adequate enough that endogenously generated active species may be
utilized as substrates in (e.g.) antimicrobial defense and xenobiotic
metabolism.

TABLE V. Clinical
trials with SOD.

SOD in its human
pharmaceutical preparation "Ontosein" has been reported to
be effective in both double blind placebocontrolled and open studies
covering a variety of human diseases. While these findings are
supportive of a role for superoxide radical in the etiology of such
diseases, they are certainly not conclusive evidence. SOD may be
acting completely independently of its ability to destroy superoxide
radical, for example, by increasing peroxide fluxes"'"
49,51,139 or even by acting directly as an immunomodulator (153).

Thus, acute free radical pathology
should occur under conditions of extraordinary radical flux. These
include inflammation, radiation or oxygen therapy, metabolism of
specific xenobiotics, or overload of autooxidationcatalyzing agents
such as manganese, copper, or iron. Further, as implied in Figure 4,
oxygen radical production may be a fundamental mediating component of
the secondary systemic response to injury ("inflammation")
from whatever source, be it (e.g.), thermal injury, drugs, or
infection.

This apparent role of oxygen
metabolites in the final common pathway of the secondary response to
tissue injury even further confounds the exact role of such species
in pathogenesis. For example, are the ameliorative effects of SOD or
catalase on the toxicity of radical-producing antitumor agents such
as adriamycin or radiation due to the inhibition of the primary toxic
action of such agents or to inhibition of the secondary systemic
response to such injury? We note that SOD may be protective even in
the case of antitumor agents for which there is no evidence of
radical production. (187)

Of great importance is providing a
basis for understanding present and potential therapy. Orgotein
("Ontoseinr'), the pharmaceutical preparation of SOD, has long
been used in veterinary medicine as an antiinflammatory agent under
the trade name "Palosein'(168-169). Indeed, orgotein was used
as an antiinflammatory agent well before it was realized it was SOD!
(Table I). This agent has recently completed clinical trials for a
variety of diseases (Table V) and currently awaits approval for human
use in the U.S. Presently its clinical use is permitted in Europe and
Japan. Perhaps the most noteworthy aspect of the pharmaceutical SOD
preparation is its low order oftoxicity. If ectopic SOD lives up to
its expectations, it will be a welcome addition to the therapeutic
armamentarium, perhaps partially replacing steroids in long-term
therapy of immunologically-based diseases such as lupus or rheumatoid
arthritis.

Potential applications of free radical
research include the use of catalase or catalase-like agents in
(e.g.) inflammatory lesions,(195), oxygen toxicity,(139),
burn-induced plasma volume loss, paraquat poisomng and immune injury
to the lung.(217). Similarly, SOD, catalase and other antioxidants
apparently ameliorate the toxicity of antitumor chemotherapeutic
agents and could be used as adjuvants to radiation therapy; while
increased understanding of the molecular basis of the toxic
potentials of other agents such as halothane and amino glycoside
antibiotics should result in ways to abrogate their toxicity.
Finally, the emerging picture of radical mechanisms as fundamental
common components of many basic pathophysiologic processes (such as
in cerebral edema, parkinsonism, and pulmonary disease) clearly has
clinical implications far beyond inflammation and drug toxicity.

Glossary

Auto-oxidation. A non-enzymatic
oxidation-reduction reaction involving two or more chemical compounds
one of which is usually oxygen or one of its active species.

Charge-transfer agent. Any
compound which can easily donate or accept electrons.

Diamagnetic. Compounds with
pairs of orbital electron(s) with paired spins, such that the
molecule has no net magnetic moment.

Dismutation. An
oxidation-reduction reaction involv-

ing a single chemical compound.

Free Radical. An atom or
molecule which has one or more electron(s) with unpaired spin(s).

Paramagnetic. Compounds with an
orbital electron of unpaired spin having a net magnetic moment.

Reduction/ Oxidation - (Redox).
Respectively, gain or loss of electrons by an ion or molecule.

Singlet state. State in which
all spin-derived magnetic fields are paired. Ground state of most
molecules, but excited state of oxygen. The form of 102 responsible
for most of its biological activity is the "Delta" form, a
potent electrophile.

Spin - Classically, the magnetic
property of the electron derived from its nature as a "spinning"
negative charge analogous to a current flowing in a "ring".
Thus, electron may be viewed as a bar magnet with its north pole
oriented either "up" or "down".

Triplet (diradical) state -
Species with even number of electrons,. but with two unpaired
electrons of parallel spin in separate orbitals. Usually excited
state, but is the ground state of oxygen.

My coauthor on this review, Edward
Reynolds, died suddenly in November of 1983. Dr. Reynolds was
chairman of the Department of Pathology at the University of Texas
Medical Branch in Galveston. He was also a major figure in the area
of the free radical-mediated toxicity of drugs, having done much of
the early work on carbon tetrachloride and halothane. Significantly,
shortly before his death, he was branching out to the area of NMR,
looking for new worlds to explore. Lt is thus fitting that this, one
of his final works, should be published in the journal that published
much of the original work on NMR.

Supported in part by a grant AM-27135
from the National Institutes of Health and by Grants from the Retina
Research Foundation, Houston, and the Alexander Medical Foundation,
San Carlos, California.

“The possibility that cerebral
ischemia may initiate a series of pathological free radical reactions
within the membrane components of the CNS was investigated in the
cat. The normally occurring electron transport radicals require
adequate molecular oxygen for orderly transport of electrons and
protons. A decrease in tissue oxygen removes the controls over the
electron transport radicals, and allows them to initiate pathologic
radical reactions among cell membranes such as mitochondria.
Pathologic radical reactions result in multiple products, each of
which may be present in too small a concentration to permit their
detection at early time periods. It is possible to follow the time course, however, by the decrease of a major antioxidant as it is consumedby the pathologic radical reactions. For this reason, ascorbic acid was measured in ischemic and control brain following middle cerebral artery occlusion. There
was a progressive decrease in the amount of detectable ascorbic acid
ranging from 25% at 1 hour to 65% at 24 hours after occlusion. The
reduction of this normally occurring antioxidant and free radical
scavenger may indicate consumption of ascorbic acid in an attempt to
quench pathologic free radical reactions occurring within the
components of cytomembranes.”